Sepsis is a whole-body inflammatory response to infection that affects approximately 1 million people each year in the United States and kills up to 50 percent of severely affected patients. Rapid and appropriate antimicrobial administration is critical for the management of sepsis. Each hour of treatment delay is associated with an 8% decrease of survival in septic shock patients. Blood culture is the gold standard diagnostic method, however, it takes more than one day to provide results. Therefore, patients receive broad-spectrum antibiotics or may not receive treatment in the initial hours. Molecular detection is an attractive solution, however, currently available tests are based on PCR amplification of bacterial genomic DNA followed by complex detection strategies. These techniques suffer from DNA contamination, lack sufficient sensitivity and are expensive. Here, we propose the development of a novel platform for rapid and inexpensive identification of microorganisms in whole blood without blood culture. The new platform will be based on Single Molecule Scanning (SM-Scanning), a novel molecular detection method in which probe hybridization to a target nucleic acid is detected without PCR amplification (Patent Pending). SM-Scanning uses a straight forward and inexpensive detection strategy. Preliminary results demonstrate that SM-Scanning can detect 5 Escherichia coli cells. In this proposal, we will demonstrate the properties of SM-Scanning by developing an assay capable of detecting microorganisms in blood samples in less than 1.5 hours with extremely high sensitivity. Aim 1 focuses on developing a rapid test capable of detecting the presence of bacteria in 5 ml whole blood samples with high sensitivity. Aim 2 focuses on developing and determining the performance of a multiplex assay capable of detecting bacteria and fungi in a single assay.